Florian Gunzer

Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.

ABSTRACT In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.

Comparison of a Shiga Toxin Enzyme-Linked Immunosorbent Assay and Two Types of PCR for Detection of Shiga Toxin-Producing Escherichia coli in Human Stool Specimens

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of sporadic cases of disease as well as serious outbreaks worldwide. The spectrum of illnesses includes mild nonbloody diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. STEC produces one or more Stxs, which are subdivided into two major classes, Stx1 and Stx2. The ingestion of contaminated food or water, person-to-person spread, and contact with animals are the major transmission modes. The infective dose of STEC may be less than 100 organisms. Effective prevention of infection is dependent on rapid detection of the causative bacterial pathogen. In the present study, we examined 295 stool specimens for the presence of Stx-producing E. coli by three different methods: an Stx enzyme-linked immunosorbent assay, a conventional PCR assay, and a LightCycler PCR (LC-PCR) assay protocol recently developed by our laboratory at the Institute of Medical Microbiology at Hannover Medical School. Our intent was to compare these three methods and to examine the utility of the STEC LC-PCR protocol in a clinical laboratory. The addition of a control DNA to each sample to clearly discriminate inhibited specimens from negative ones enhanced the accuracy of the LC-PCR protocol. From our results, it can be concluded that LC-PCR is a very useful tool for the rapid and safe detection of STEC in clinical samples.

The host response to the probiotic Escherichia coli strain Nissle 1917: Specific up-regulation of the proinflammatory chemokine MCP-1

BackgroundThe use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells.MethodsGene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins.ResultsWhole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2α) and macrophage inflammatory protein-2 beta (MIP-2β) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2α mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue.ConclusionThus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells.

Gnotobiotic Piglets Develop Thrombotic Microangiopathy After Oral Infection With Enterohemorrhagic Escherichia coli

Oral infection with enterohemorrhagic Escherichia coli (EHEC) may cause severe enteritis, followed in up to 10% of cases by an extraintestinal complication, the hemolytic uremic syndrome (HUS). HUS is characterized by a triad of symptoms: anemia, thrombocytopenia, and acute renalfailure due to thrombotic microangiopathy. EHEC produces several virulence factors, among which a family of phage-encoded cytotoxins, called Shiga toxin 1 and Shiga toxin 2, seems to be most important. However, since an appropriate animal model is not available, pathogenicity of these emerging enteric pathogens is still poorly understood. Germ-free gnotobiotic piglets infected orally with an O1577:H7 or an O26:H11 EHEC wild-type isolate, both producing Shiga toxin 2, developed intestinal and extraintestinal manifestations of EHEC disease, including thrombotic microangiopathy in the kidneys, the morphologic hallmark of HUS in humans. Thus, gnotobiotic piglets are suitable to further study the pathophysiology of EHEC-induced HUS. It can be expected that data obtainedfrom this animal model will improve our current standard of knowledge about this emerging infectious disease.

Candida kefyr as an emerging pathogen causing nosocomial bloodstream infections in neutropenic leukemia patients

Figure 1. Response of Candida kefyr bloodstream infection in patient 3 to combination antifungal therapy that included caspofungin. Staphylococcus epidermidis was also detected in 2 blood cultures but disappeared after antibiotic treatment, according to the antibiogram. Day 0 was the day of allogeneic peripheral blood stem cell transplantation (PBSCT). a, Graph depicting recovery of the leukocyte and the granulocyte counts. b, Serum levels of C-reactive protein (CRP). +, Blood cultures positive for C. kefyr. *Blood cultures positive for S. epidermidis. c, Graph showing the highest daily body temperature. d, Schematic representation of the antifungal therapy given after PBSCT. Dosages were as follows: nystatin, 1.5 million IU/day orally; caspofungin, 50 mg/day intravenously; fluconazole, 600 mg/day intravenously; amphotericin B, 3 mg/kg/day intravenously. 12. Kontoyiannis DP, Lionakis MS, Lewis RE, et al. Zygomycosis in a tertiary-care cancer center in the era of Aspergillus active antifungal therapy: a case control observational study of 27 recent cases. J Infect Dis 2005; 191:1350–60.

Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli

BackgroundEnterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors.MethodsUsing rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance.ResultsFirst, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant.ConclusionThe attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.

Therapy of solid tumors using probiotic Symbioflor-2 – restraints and potential

To date, virulent bacteria remain the basis of most bacteria mediated cancer therapies. For clinical application attenuation is required. However, this might result in a drastically lowered therapeutic capacity. Herein we argue that the E. coli probiotic Symbioflor-2, with a history of safe application may constitute a viable tumor therapeutic candidate. We demonstrate that Symbioflor-2 displays a highly specific tumor targeting ability as determined in murine CT26 and RenCa tumor models. The excellent specificity was ascribed to reduced levels of adverse colonization. A high safety standard was demonstrated in WT and Rag1−/− mice. Thus, Symbioflor-2 may represent an ideal tumor targeting delivery system for therapeutic molecules. Moreover, Symbioflor-2 was capable of inducing CT26 tumor clearance as result of an adjuvant effect on tumor specific CD8+ T cells analogous to the Salmonella variant SL7207. However, lower therapeutic efficacy against RenCa tumors suggested a generally reduced therapeutic potency for probiotics. Interestingly, concurrent depletion of Gr-1+ or Ly6G+ cells installed therapeutic efficacy equal to SL7207, thus highlighting the role of innate effector cells in restraining the anti-tumor effects of Symbioflor-2. Collectively, our findings argue for a strategy of safe strain application and a more sustainable use of bacteria as a delivery system for therapeutic molecules.

Development of a Rapid Microarray-Based DNA Subtyping Assay for the Alleles of Shiga Toxins 1 and 2 of Escherichia coli

ABSTRACT In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise.